An increase in cellular protein misfolding and a decrease in degradation of proteins are characteristics of cellular and organismal aging. Prevention of misfolded protein accumulation is normally mediated by the cellular quality control (QC) machinery, including chaperones and the proteasome. Loss of QC machinery function can occur at multiple regulatory levels. However, many of the QC components are especially long lived proteins. Acute loss of a cell's QC capacity predisposes the cells to rapid death. In contrast, aging appears to be a gradual process that occurs over the course of several days to years, not a catastrophic process. We hypothesize that discrete components of the cellular protein quality control and homeostasis machinery are selectively and progressively inactivated during aging, potentially due to oxidative damage. As a consequence, the ability of cells to respond to misfolded protein stress or maintain homeostasis is likely to be impaired. While studies have addressed changes in mRNA levels, protein levels, and oxidative damage to QC proteins, little is known regarding the functionality of the QC proteins in aging cells. In this proposal, we will directly measure QC protein function at the levels of individual QC proteins, their substrates, and global misfolded protein stress. These problems will be investigated by 1) developing new methods for detecting misfolded protein stress and QC machinery function in living cells, 2) determining which QC components lose functionality in aging cells and in an aging model of oxidatively stressed cells, and 3) using genetic, pharmacologic, and biophysical fluorescence methods to define how crosstalk between cytoplasmic and secretory protein QC systems modulate misfolded protein levels and stress in aging cells. Project Narrative: Several protein misfolding diseases increase in incidence and severity as people age. We are studying, at the cellular level, the mechanisms that normally prevent misfolded proteins from accumulating in cells and how these mechanisms are altered during aging.